In general, since PDI has a capability of catalyzing a disulfide exchange in proteins, the application thereof to, for example, refolding of recombinant proteins produced by utilizing, as a host, prokaryotic cells, such as Escherichia coli and Bacillus subtilis, have been studied in the art. The term "refolding" used herein is intended to mean that proteins incapable of exhibiting an inherent physiological activity due to erroneous bonding form of a disulfide bond are converted to an active type by correcting the bonding form of the disulfide bond.
Examples of conventional methods for refolding the recombinant protein inactive due to a deficiency of the disulfide bond include a method wherein use is made of a chemical redox reaction (see Japanese Unexamined Patent Publication (Kokai) No. 1-131195) and a method wherein use is made of PDI (see Japanese Unexamined Patent Publication (Kokai) No. 63-294796). Since PDI has been considered as an enzyme actually involved in the formation of a disulfide bond of proteins in vivo, the utilization of PDI in a reaction in vitro is a very rational conception.
Since PDI was first found by Anfinsen et al. as an enzyme capable of catalyzing the formation of a disulfide bond (see J. Biol. Chem., 238, 628 (1963)), PDI is known to be widely distributed in several tissues of mammals and to exhibit a high activity particularly in organs wherein proteins having a disulfide bond are actively synthesized or secreted (such as liver, pancreas, spleen and lymphatic tissue) (see Hillson, D. A., Lambert, N., Freedman, R. B., Methods in Enzymology, 107, 281-294, 1984). In recent years, it has been found that PDI is present in, besides mammals, green algae (see Kaska, D. D., Kivirikko, K, I., Myllylae, R.: Biochemical Journal, 268, 63-68, 1990) and yeast (see Mizunaga, T., Katakura, Y., Miura, T., Marugama, Y., Journal of Biochemistry, 108, 846-851, 1990). Known features common to these PDI's are such that they has a molecular weight of 52,000 to 62,000, comprise a dimer comprising identical two subunits and an isoelectric point of 4.0 to 4.5. Further, the amino acid sequence of PDI has a feature that it has two sequences of (Trp-Cys-Gly-His-Cys-Lys; SEQ ID NO:4) considered as an active site in a subunit polypeptide chain.
PDI's per se and techniques for utilizing PDI in refolding of proteins have already been reported in the art (see Japanese Unexamined Patent Publication (Kokai) Nos. 4-197176; 2-460, 64-20086 and 63-294796). The PDI's used therein are derived from mammals and yeast, and have a problem of stability when they are used in the above-described purposes. For this reason, it is necessary to provide a polypeptide possessing PDI activity which has a significantly higher stability than the conventional PDI, can be used in a wide range of temperatures and is resistant to a sulfhydryl-group-containing reducing agent in a wide concentration range generally necessary to be added in a refolding reaction system, for example, dithiothreitol (hereinafter abbreviated to "DTT"). Further, the provision of such an active protein in turn leads to a desire for the development of a technique for mass-producing the active protein.
Accordingly, the first object of the present invention is to provide a polypeptide possessing PDI activity having a higher stability than conventional PDI's. The second object of the present invention is to provide a production process which enables PDI having a high stability to be mass-produced with a high efficiency.